(Received 7 April 1967)

Transcription

1 J. Phy8iol. (1967), 192, pp With 16 text-ftgure8 Printed in Great Britain THE RESPONSES OF THALAMIC NEURONES TO IONTOPHORETICALLY APPLIED MONOAMINES By J. W. PHILLIS AND A. K. TEBRCIS From the Department of Physiology, Monash University, Clayton, Victoria, Australia (Received 7 April 1967) SUMIARY 1. The effects of noradrenaline (NA), adrenaline, dopamine (DA) 5-hydroxytryptamine (5-HT) and a number of related drugs were tested on the extracellularly recorded responses ofneurones in the feline thalamus. Substances were applied iontophoretically and tested on synaptically, antidromically and chemically evoked neuronal activity. 2. NA, adrenaline, isoprenaline and 5-HT had a variety of effects, depressing some cells, exciting others and not affecting the responses of a third group. DA depressed most of the cells tested; excitation was not observed with this compound. 3. The magnitude of depressant actions and their duration varied considerably. The more sensitive cells responded to extremely small amounts of catecholamine or 5-HT and recovery often took several minutes. Recovery after DA was always rapid. Neurones in the dorsal thalamus were generally more susceptible to depression than those in the ventro-basal complex. 4. Excitatory responses were most marked in the ventro-basal complex of the thalamus. Desensitization occurred if NA or adrenaline was applied repeatedly and this tachyphylaxis lasted for several minutes. After desensitization to the excitatory effects, some of these cells were depressed by catecholamines. These findings suggest the presence of at least two types of membrane receptor. 5.,J-adrenergic antagonists (alderlin, D-INPEA and MJ 1999) and a-antagonists (phentolamine, dibenzyline and chlorpromazine) had pronounced depressant actions on some thalamic neurones. With the exceptions of D-INPEA and MJ 1999 they also excited cells that were excited by the catecholamines. Alderlin and phentolamine had both excitatory and inhibitory effects on some cells. 6. The monoamine oxidase inhibitor, iproniazid, depressed neurones which were sensitive to NA depression. It did not appear to potentiate the effects of NA on most of the cells tested.

2 716 J. W. PHILLIS AND A. K. TEBECIS 7. Reticular formation stimulation depressed some neurones in the thalamus and excited others. The depressant effects of NA and reticular formation stimulation were reduced or abolished by an intravenous injection of picrotoxin (1 mg/kg). 8. It is suggested that NA and 5-HT may be inhibitory transmitters in the thalamus, released at the terminals of ascending pathways from the brain stem that have been defined by fluorescence microscopy. The excitatory actions of these compounds may also be related to a synaptic role. INTRODUCTION The biogenic monoamines, 5-HT, DA and NA are widely distributed throughout the central nervous system. Although their functional significance is still uncertain, a definite association with specific anatomical systems has now been established by the use of biochemical and histochemical techniques. The biochemical method involves the unilateral destruction of specific fibre tracts in the brain, such as the medial forebrain bundle, and subsequent comparisons of the amine contents of the operated and control sides of the brain. Unilateral lesions in the lateral hypothalamus of the cat cause a fall in the NA and 5-HT content of the telencephalon on the ipsilateral side of the brain (Moore, Wong & Heller, 1965; Heller, Seiden & Moore, 1966). In the rat, lesions of the medial forebrain bundle also cause a significant fall in the brain monoamine levels, including DA, on the side of the lesion (Harvey, Heller & Moore, 1963; And6n, Dahlstr6m, Fuxe, Larsson, Olson & Ungerstedt, 1966). The existence of catecholamine- and 5-HT-containing nerve cells and fibres in the rat and cat brain has also been established by means of a histochemical fluorescence method (Falck & Owman, 1965) and there is evidence that the monoamine-containing nerve terminals in the brain are true synaptic terminals, containing high amine concentrations (Fuxe, 1965a, b). A tentative schematic outline of ascending monoaminergic projections in the rat brain, based on evidence obtained by both of the techniques mentioned, has recently been presented by And6n et al. (1966). This outline includes both noradrenergic and 5-hydroxytryptaminergic projection systems from the medulla and pons to the thalamus. NA, DA, and 5-HT are present in the feline thalamus (Bogdanski, Weissbach & Udenfriend, 1957; Bertler & Rosengren, 1959; McGeer, McGeer & Wada, 1963), the levels of NA in the cat being similar to those in the comparable area of rat brain (Glowinski & Iversen, 1966). Fine, varicose, NA and 5-HT containing nerve fibres have been observed in the central nervous system of cats by fluorescent histochemical techniques. The distribution of these fibres is apparently comparable in cats and rats

3 MONOAMINES ON THALAMIC NEURONES 717 (Fuxe, 1965a) and it is therefore reasonable to assume that monoaminecontaining synaptic terminals are present in the feline thalamus. The present report concerns the actions of monoamines and related compounds on cells in dorsal and ventral areas of the feline thalamus. A subsequent paper will deal with the actions of cholinergic compounds on thalamic neurones. The papers form part of a sequence dealing with the actions of monoamines and cholinesters on neurones in the central nervous system (Krnjevi6 & Phillis, 1963 a, b, c; Phillis, Teb6cis & York, 1967a, b). METHODS Adult cats were anaesthetized with intravenous thiopentone sodium (Intraval Sodium, May & Baker), supplementary doses of whichwere administered as required until the preparation of the animal had been completed. Anaesthesia was maintained with nitrous oxide, supplemented with either halothane (Fluothane, I.C.I.) or methoxyflurane (Penthrane, Abbott). The head of the animal was fixed in a stereotaxic frame and the abdomen rested on an automatically controlled heating pad, which maintained the preparation at C (Krnjevi6 & Mitchell, 1961). After removal of the appropriate parts of the skull, cortical and subcortical tissue of the left cerebral hemisphere was removed by suction to expose the hippocampal fornix and fimbria and the floor of the lateral ventricle between the stereotaxic co-ordinates A7 to A 12 and L 0 to L 9. The rostral border of the cortical ablation was restricted to avoid excessive damage to thalamo-cortical fibres to the somatosensory areas I and II. The hippocampus, fornix and fimbria were left in situ to miimize damage to the vascular supply to the dorsal thalamic nuclei. Two glass micropipettes were inserted stereotaxically to a depth of 10 mm below the surface of the fornix, cut at a point 2 mm above the brain surface and left in position. These acted as reference points for the alignment of new electrodes and were useful markers for the selection of blocks of tissue when brains were being prepared for histology. The left somatosensory cortex was exposed and five bipolar concentrio stimulating electrodes inserted into the pericruciate area and somatosensory cortex number II. These were adjusted so that their tips were located about 4 mm below the cortical surface and used to stimulate the terminal portions of thalamocortical fibres in order to activate antidromically neurones in the ventrobasal complex of the ipsilateral thalamus. The exposed cortical surface was covered with a 2-3 mm thick layer of 4% agar in physiological saline as previously described (Phillis et al. 1967a). In some experiments, the superficial radial, median and ulnar nerves were exposed and mounted on bipolar stimulating electrodes in a paraffin pool made of skin flaps. On other occasions stainless.steel pins were inserted into the extremities of the digits and used for stimulation of limb afferent nerves. Bipolar concentric stimulating electrode was placed on the ipsilateral brachium conjunctivum under direct vision after the cerebellar cortex had been sucked away in the mid line. Other bipolar stimulating electrodes were placed stereotaxically in the mesencephalic reticular formation and centrum medianum. Evoked field potentials and spike potentials were recorded through a sodium chloride (2M) filled barrel of a multiple barrelled micropipette. This recording barrel was connected to a negative capacitance probe (Bioelectric Instruments) and potentials were displayed on a Tektronix 565 oscilloscope after preamplification, and in some instances recorded on film. The output of the preamplifier was also connected in parallel to a second oscilloscope, two

4 718 J. W. PHILLIS AND A. K. TEBECIS spike intensifiers (Kellett, Phillis & Veale, 1965) and an audio amplifier. The second oscilloscope was used to monitor the output pulses of one of the spike intensifiers which was connected to an electronic counter (Hewlett Packard 5214L). This spike intensifier operated as a variable voltage gate and pulse generator, enabling the rejection of all input signals smaller than those of the unit primarily under observation. It also provided the counter with a uniform series of pulses corresponding to the cell discharges. Gate times on this counter range from 10,usec to 100 sec, but it was most frequently used with gate times of sec. The output of the counter was displayed on an ink recorder (Texas Instruments Recti-Riter) using an analogue output coupling stage of a Hewlett Packard 562A digital recorder. The construction and filling of multiple-barrelled micro-electrodes has already been described in detail (Krnjevi6 & Phillis, 1963a; Phillis et al. 1967a). Aqueous solutions of the various monoamines were made up at a ph of 35-5 to improve their stability and the filled electrodes were stored in the dark at 40 C. One barrel of each nine-barrelled and of some of the five-barrelled electrodes was filled with Ol1N hydrochloric acid for making lesions (McCance & Phillis, 1965). Drugs were applied as cations, with the exception of L-glutamate. A retaining potential was applied to all drug containing barrels to prevent the diffusion of actively ionized compounds. At the termination of those experiments in which acid lesions had been made in the thalamus, the animals were perfused with 0.9% saline followed by 10% formol saline. After further fixation, 50 /% serial sections of the thalamus were cut on a freezing microtome and stained with Luxol Fast Blue and Neutral Red (Lockard & Reers, 1962). Photomicrographs of whole sections were taken with a Nikon 6 C Shadowgraph. RESULTS The results described in this paper concern the actions of monoamines and related compounds on nerve cells distributed over several thalamic nuclei. In many instances the precise position of neurones could be confirmed by histological examinations of the thalamus and identification of the sites of 'acid lesions' (McCance & Philhis, 1965) (see Figs. 1 and 2). When an HCl-containing barrel was not included in a micro-electrode, the position of cells in the thalamus had to be estimated from stereotaxic co-ordinates. Comparisons of the location of lesions with the co-ordinates at which the tracks were placed have confirmed that reliance can be placed on the latter method of assessing the position of cells. The most frequent hazard involved in this stereotaxic method resulted from brain swelling during the latter stages of experiments. Other criteria for identifying the position of cells included their ability to respond orthodromically to stimulation of the brachium conjunctivum (BC) or antidromically to stimulation of the sensori-motor area of the cerebral cortex. Responses to either of these stimuli indicate that the cells are likely to be in the ventrolateral complex of the thalamus (McCance, Phillis & Westerman, 1967). On account of the considerable difficulties involved in collating evidence in a multidimensional relationship, the results have been related to the depth of cells below the uppermost extent of the thalamus. Laterality and

5 MONOAMINES ON THALAMIC NEURONES anteroposterior dimensions have been omitted from the analysis, as they did not appear to influence the results significantly. Neurones have been grouped into three depth categories: superficial group cells at depths of 0-3 mm; intermediate group at depths of 3-6 mm; deep group cells at depths of 6-9 mm. Thalamic nuclei included in the depth boundaries of the Superficial Group are: N. anterior dorsalis and ventralis, N. lateralis dorsalis, N. centralis lateralis, N. ventralis anterior, N. medialis dorsalis. The Intermediate Group includes: N. centralis lateralis, N. lateralis posterior, N. medialis dorsalis, N. ventralis lateralis, N. ventralis anterior, N. reticularis. The Deep Group area includes N. ventralis lateralis, medialis, posteromedialis and posterolateralis and N. centralis medialis. Noradrenaline NA was tested on 227 cells in nineteen cats. The results are summarized in Table 1. An approximately constant proportion of the cells tested were unaffected by NA (in the amounts applied) at all three depth levels. TABLE 1. Actions of noradrenaline on thalamic neurones* 719 Action Superficial Intermediate Deep No effect 26 (34%) 31 (45%) 27 (33%) Depression 48 (630%) 24 (350/ ) 30 (37%) Excitation 2 (30%) 14 (20o0 ) 25 (30%) * In this and subsequent tables the figures indicate numbers of neurones tested in each depth group and the responses observed. Depression was the most frequently observed result with cells in the superficial group and as the electrode descended an increasing proportion of excitable cells was encountered. This disproportion with respect to the effects of NA on cells at different depth levels is statistically significant (P < 0.01). The phenomena of NA depression and excitation can most conveniently be described independently. NA-depression. Many of the cells tested with NA were located by applying L-glutamate continuously as the electrode passed through the thalamus. It was frequently possible to identify the position of these cells by the 'lesioning' technique which has been extensively employed in other experiments on thalamus, lateral geniculate nucleus and cerebellum (McCance, Phillis & Westerman, 1966; Phillis et al. 1967a; McCance & Phillis, 1964). Examples of the responses of neurones in the superficial depth group are show-n in Fig. 1. Six lesions are visible in this track, four of them being in close apposition. The usual technique adopted for testing the excitability 46 Physiol. I92

6 720 J. W. PHILLIS AND A. K. TEBECIS of such cells was to apply L-glutamate at approximately 30 sec intervals for periods of 10 sec. When the current through the L-glutamate barrel had been adjusted to give repeatable responses, a cationic current was passed through the NA-containing barrel, applications of L-glutamate being continued. Depressant or excitant actions of NA are apparent as a reduction or enhancement of the rate of firing induced by L-glutamate. When neurones were also excited by ACh, it was possible to test the effects ofna on the actions of both this compound and L-glutamate. c NA 50 A NA 50 J ifl. UJ oo 100 I-ilkiuli NA 50 B NA 50 ]~~~~L j m D *-: sec 30 sec Fig. 1. Differences in depressant effects of NA (50 na) on four thalmic neurones (A, B, C and D) at depths of 650 (A), 990 (B), 2120 (C) and 2740 (D) microns. L-glutamate was applied by currents of 30 na (A), 60 na (B), 70 na (C) and 20 na (D). Positions of recording are illustrated by lesions in the photomicrograph. In this and subsequent records, periods of drug application are indicated by horizontal lines above and/or below trace. The responses of four cells to L-glutamate and NA are shown in Fig. 1 (the white darts indicating which lesion corresponds to each cell). Three of these cells were clearly depressed by NA and the fourth (D) may have been slightly depressed. Neurones in the superficial group were usually more sensitive to NA depression than those in the intermediate and deep groups. This sensitivity was manifested by the smaller NA applying currents necessary to abolish L-glutamate firing and frequently excitability recovered slowly over several minutes after an application of NA. Deeper cells were generally less affected by NA and recovery after an application was rapid. Examples of the responses of cells in the intermediate and deep groups to NA are shown in Fig. 2. Cell A is located in the intermediate depth area,

7 MONOAMINES ON THALAMIC NEURONES 721 cell B lies on the boundary between the two areas and the remaining cells are in the deep group. All of the cells represented in this figure were excited by ACh as well as L-glutamate, and these two substances were applied alternately to estimate whether their excitant actions were reduced to a comparable extent. With the exceptions of cells A and F, NA depressed cell excitability, reducing L-glutamate and ACh excitation to the same extent. Recovery was rapid in each instance. Cells C, D and F responded with short constant latency spikes to stimulation of the subcortical white matter in the sensorimotor area of the ipsilateral cerebral cortex and were therefore classified as thalamocortical units. NA 50 A - --D 0~~~ iii 1111I GGA GAGA NA SO 3 mm E NA 50 GGA GA GA 200I c C NA 60 f~--- NA 80 GA GAGAG G A GA 60 F 200 NA! 200[.ih...1~~~~IL GAG GA20 LLIL.LJ*~~~~~~LL-.LL 30 sec 30 sec Fig. 2. Differences in depressant effects of NA (50-80 na) on L-glutamate and ACh induced firing in six cells of the intermediate and deep groups. Positions are indicated by lesions in the photo mierographs. ACh (A) was applied by currents (in na) of 20 (A), 20 (B), 12 (C), 20 (D), 15 (E) and 30 (F). L-glutaMate (G) was applied by currents (in na) of 40 (A), 20 (B), 20 (C), 20 (D), 20 (E) and 30 (F). NA depression of both ACh and L-glutamate excitation indicates that it caused a reduction in neuronal excitability, rather than a specific block of the actions of either excitant compound. This conclusion was supported by the finding that NA also depressed synaptic activation of sensitive neurones. Stimulation of the ipsilateral brachium conjunctivum (BC) evokes a monosynaptic excitatory response in neurones in the area of the ipsilateral thalamus to which this pathway projects (McCance et al. 1967). 46-2

8 722 J. W. PHILLIS AND A. K. TEBECIS A NA rn (4) B I mv msec C NA 50 GA GA GA -200 U 100 ajn _! CL E 0O Min Fig. 3. Depressant effects of NA (50 na) on a thalamo cortical unit recorded in the deep layer. This unit was evoked antidromically by stimulation of the ipsilateral sensori-motor cortex (A) and orthodromically from the brachium conjunctivum (B). (Eachpair of photographs inbrepresents consecutive records.) Controlresponses are shown in A (1) and B (1). NA (50 na) depressed the orthodromic response B (2) but not the antidromic response A (2). A (3) and B (3) were recorded several minutes after termination of NA application. A (4) and B (4) represent responses to repetitive stimulation. A (4), 100/sec cortical stimulation. B (4, first record), 30/sec and B (4, second record), 10/sec BC stimulation. C is simultaneous servo/riter record illustrating depression ofl-glutamate (50 na) and ACh (50 na) firing by NA (50 na).

9 MONOAMINES ON THALAMIC NEURONES 723 The constancy of this response renders it particularly suitable for use in testing the actions of pharmacological agents on synaptic transmission. A typical monosynaptic response to BC stimulation is shown in Fig. 3B (1). This neurone also responded with a short latency spike to stimulation of the ipsilateral sensorimotor cortex (Fig. 3A (1)), but whereas it followed cortical stimulation at frequencies in excess of 100/sec, it failed to follow BC stimulation at frequencies in excess of 10/sec. NA (50 na) application resulted in a failure of the synaptically evoked response (Fig. 3B(2)) although the antidromic spike continued to invade the cell Fig. 3A (2)). Responses to BC stimulation recovered rapidly after termination of the NA application. NA also reduced the excitant actions of ACh and L-glutamate, the trace in Fig. 3 0 being recorded at the same time as those in Fig. 3A and B. Large NA currents (in excess of 100 na) caused a partial TABLE 2. Actions of adrenaline, isoprenaline and dopamine on thalamic neurones Action Superficial Intermediate Deep Adrenaline No effect 22 (360/) 19 (34%) 7 (30%) Depression 35 (55) 32 (570) 14 (61%) Excitation 6 (90) 5 (90) 2 (90) Isoprenaline No effect 8 (53%) 6 (26%) 6 (43%) Depression 6 (400%) 14 (61%) 5 (36%) Excitation 1 (70) 3 (13%) 3 (21%) Dopamine No effect 4 (14%) 0 1 (11%) Depression 24 (86%) 11 (100%) 8 (89%) Excitation failure of invasion of antidromic spikes into the neurone soma. The invasion of antidromic spikes into the soma of other thalomocortical neurones was difficult to abolish with NA. This finding is not incompatible with the suggestion that NA is depressing neuronal excitability, as the safety margin for the antidromic propagation of spikes into thalamocortical neurones may be relatively high. 5-HT, which is a potent depressant of lateral geniculate neurones, failed to prevent the invasion of an antidromic spike into the soma of some neurones in this nucleus (Phillis et al a) and y-aminobutyric acid was less effective in abolishing antidromically evoked spikes of Betz cells in the cerebral cortex than spikes evoked by chemical or synaptic stimulation (Krnjevic6 & Phillis, 1963d). A similar explanation is likely to be valid in these latter instances. Depression by dopamine, adrenaline and isoprenaline. Dopamine was tested on forty-eight cells, adrenaline on 142 cells and isoprenaline on fifty-two cells. The results are summarized in Table 2. Adrenaline and isoprenaline were frequently more active than NA as depressants, but their actions were otherwise identical. The depressant effects of the three compounds are presented in Fig. 4. L-glutamate was used to test the

10 724 J. W. PHILLIS AND A. K. TEBECIS excitability of both cells during consecutive applications of adrenaline, isoprenaline and NA. Adrenaline and isoprenaline were considerably more active as depressants on both cells. Both substances also depressed the synaptic activation of thalamic neurones during BC stimulation in an analogous manner to NA. A A 60 NA 60 Isopren j50w E _o m ~6 B lsopren 40 A 40 NA _ ~- _ Fig. 4. Depressant effects of three catecholamines on two cells in the deep layer. A (6470,u), B (8940,). In both cases adrenaline (A) and isoprenaline were more potent than NA (applied by equal currents). Applications of L-glutamate (30 and 40 na for A and B, respectively) are indicated by horizontal lines below the responses. DA depressed the excitability of most of the neurones on which it was tested, and its action also appeared to differ from that of NA in other respects. A typical example of the action of DA on a NA sensitive neurone is shown in Fig. 5. DA (20 na) caused a marked reduction in the excitability of the cell (which was in the superficial group) as evidenced by the large reduction in L-glutamate evoked firing, Recovery occurred within a few seconds of the termination of dopamine application. NA (20 na), on the min

11 MONOAMINES ON THALAMIC NEURONES 725 other hand, caused a comparable reduction in the response to L-glutamate and recovery was slow. Four minutes elapsed before the excitability of the cell returned to its normal level. DA also depressed the synaptic activation of cells during BC stimulation. An excitant action ofda was never observed. DA 20 NA Fig. 5. A comparison of depression induced by DA (20 na) and NA (20 na) in a cell 1740 /s below the surface ofthe thalamus. L-glutamate applications were by currents of 30 na. DA caused depression followed by a typically rapid recovery. NA caused depression followed by a slow recovery, which was frequently observed with cells of the superficial group. Excitation by noradrenaline, adrenaline and isoprenaline. The excitant actions of the catecholamines, other than dopamine, have already been mentioned in Tables 1 and 2. Most of the results have been obtained with NA, which excited a greater proportion of the cells on which it was tested than the other catecholamines. The marked variability of the excitant actions of these compounds on cells in different animals, coupled with the phenomenon of desensitization with repeated applications, has made it difficult to compare their excitant potencies. Excitation of some neurones has been observed in all the animals tested with these catecholamines, although the effects were much more definite in some instances. Excitation was most consistently observed when the catecholamines were tested on thalamocortical neurones in the ventro-basal nuclei. An example of such a cell is shown in Fig. 6. This neurone responded with an antidromically invading spike when the sensori-motor cortex was stimulated on the ipsilateral side (Fig. 6A) and followed cortical stimulation at frequencies of 100/sec. It was typically sensitive to ACh excitation and responded to L-glutamate (20 na). NA (20 na) also excited the cell, its effects having a somewhat slower onset and offset than those of the other two excitants (Fig. 6B). The excitability of other cells in the thalamus was min

12 726 J. W. PHILLIS AND A. K. TEBECIS increased by NA to a limited extent only. This action was evident when the cell was tested with L-glutamate or ACh during or immediately after the catecholamine application, an increase in the response frequency being observed. An example of this type of response is shown in Fig. 15B. When NA or adrenaline was applied repeatedly to the same neurone it rapidly became less sensitive to their excitant actions. Examples of this desensitization are presented in Figs. 7 and 8. The thalamocortical unit in Fig. 7 responded to cortical stimulation and was excited by L-glutamate, ACh and NA (Fig. 7 b). NA was then applied repeatedly, with progressively less effect, until by the fourth application it had no effect, even though the applying current had been increased from 30 to 50 na. The constancy of the L-glutamate responses indicates that the excitability of the unit had Fig. 6. Excitatory effects Of L-glutamfate (20 na), ACh (20 na) and NA (20 na) on a thalamocortical unit in the ventro basal nucleus. A: antidromic response to stimulation of the sensori motor area of the ipsilateral cortex. B: oscilloscope records of spikes evoked by application of drugs. Excitation induced by NA usually had a longer latency of onset and offset in comparison with ACh and L-glutamate. not been altered.- When NA was tested again after a 10 min interval it had quite a marked excitant action. A noticeable feature of the NA excitation of several deep neurones was its long duration of action. Some of these cells which had been quiescent before the NA& application commenced to fire during it and continued to fire for several minutes subsequently. During the period of continued firing, further applications of NA had little further effect. A dual effect of NA was observed on some neurones. NA initially had an excitant action on such cells, but with repeated applications this response was replaced by a depression Of L-glutamate induced firing. A sequence of adrenaline applications is shown in Fig. 8. This unit was located in the intermediate depth group and responded to the initial application of adrenaline (30 na) with a period of marked excitation. A second application of adrenaline precipitated two isolated bursts of spikes and the third and fourth applications were almost without effect. Increasing

13 MONOAMINES ON THALAMIC NEURONES 727 the adrenaline current to 60 na did not increase its effect. After a period of several minutes the desensitization wore off and the cell recommenced to respond to adrenaline. A B NA _ mv 100Ia msec f min C - NA 30 NA 3O NA3 NA 50 NA IW L II]I min Lu 0 Fig. 7. An example of desensitization of a thalamo cortical neurone as a result of repeated applications of NA (30 and 50 na). A (top record); antidromic spike evoked by stimulation of ipsilateral sensori-motor cortex; (bottom record): same response at stimulation of 100/sec. B: same unit firing to L-glutamate (40 na), ACh (20 A) and NA (30 na). Excitation by NA had a long latency of onset and offset. C: a series of applications of L-glutamate (40 na) and NA, illustrating a decreasing excitatory effect of NA with each successive application. The fourth application of NA evoked no firing. After a 10 min rest, NA (30 na) again had a potent excitatory action on the cell. Firing induced by L-glutamate (40 na) remained virtually at the same level throughout this series.

14 728 J. W. PHILLS AND A. K. TEBECIS The generation of synaptic responses was facilitated in those neurones which were excited by NA. L-glutamate, ACh and BC stimulation had minimal effects on the thalamocortical neurone in Fig. 9. A small negative field was generated by BC stimulation, but the unit itself failed to reach threshold for the initiation of a spike (Fig. 9B (1)). During the application of NA (30 na), the neurone commenced to fire (Fig. 9 C) and the responses to ACh were greatly enhanced, even when the rate of firing had nearly returned to its control levels. The field evoked by BC stimulation was greatly enhanced and the cell commenced to respond to BC stimulation (Fig. 9B (2)). As the increase in excitability declined after the NA application, there was a reduction in the size of the evoked field, the cell ceased to respond synaptically and the ACh response returned to its control levels G 35 A 30 A 30 A 30 A 30 Fig. 8. An example of desensitization to adrenaline (30 na) of a thalamic neurone at a depth of 3810,u. The first application of adrenaline evoked a violent burst of firing which persisted for 2 min after termination of the ejecting current. Successive applications produced less effect. The fourth application produced almost no effect. Firing to L-glutamate (35 na) remained constant at the beginning and end of this series. Catecholamine antagonists A series of compounds which antagonize the actions ofthe catecholamines on both central and peripheral receptors has been tested on thalamic neurones. The compounds and the numbers of cells on which they were tested are: 2-isopropylamino-l-(2-naphthyl) ethanol hydrochloride (alderlin, pronethalol) forty-two cells; 2-N(m-hydroxyphenyl)-N-p-tolyl-aminomethyl-2-imidazoline methanesulphonate (phentolamine) thirty-nine cells; chlorpromazine, thirty-four cells; D-, L- and DL-l-(4-nitrophenyl)-2-isopropylaminoethanol hydrochloride (INPEA) on eighteen cells; 4-(2-isopropylamino-l-hydroxy ethyl) methanesulpbonanilide hydrochloride (MJ 1999) on fourteen cells; N-(2-chloroethyl)N-(2-phenoxyisopropyl) benzylamine hydrochloride (dibenzyline) on ten cells. min

15 MONOAMINVES ON THALAMIC NEURONES 729 Fig. 9. Facilitation of a synaptic response by NA (30 na). The thalamocortical unit was recorded at a depth of 7200,t. A (top record): antidromic response to stimulation of ipsilateral cortex; (bottom record): same response following repetitive stimulation of 180/sec. B (1): consecutive records of a field evoked by stimulation of the brachium conjunctivum. B (2): spikes evoked by BC stimulation after NA (30 na) had been applied for 1 min. C: simultaneous servo-riter record illustrating excitatory effects of NA (30 na). Before application of NA, L-glutamate (40 na) and ACh (30 and 60 na, respectively) induced a relatively low firing frequency. After firing from NA had subsided, ACh (40 and 20 na, respectively) caused a high rate of firing.

16 730 J. W. PHILLIS AND A. K. TEBECIS All the catecholamine antagonists had depressant actions on some of the neurones on which they were tested. The magnitude of the depressant action usually paralleled that of the catecholamines which were present in the same electrode. Examples of the depressant actions of phentolamine and alderlin on two cells are shown in Fig. IOA, B. Both substances depressed the responses to L-glutamate and recovery after termination of applications was slow. A Phentolamine ~ 50 JLJJh~~~LLAL~~~U1jO5~0 E. B Alderlin u 50 W 0 C Alderlin 40 NA 60 Phentolamine 50 l~~~~~~~~~~~~ 300 J, ~~~~~~~~~~~~~~l Fig. 10. Examples of responses of thalamic neurones to phentolamine and alderlin A (1890 /z): phentolamine (20 na) gradually depressed firing evoked by L-glutamate (70 na). Recovery took 5 min. B (4480 /,t): alderlin (60 na) depressed L-glutamate (60 na) firing and recovery was slow. C (7100,): alderlin (40 na) had an initial excitatory action followed by a depression ofl-glutamate (50 na) firing. NA (60 na) also caused a marked increase in firing rate. Phentolamine (50 na) exhibited slight excitatory activity, manifested as an increase in rate of background firing and a potentiation of L-glutamate (50 na) firing. min

17 MONOAMINES ON THALAMIC NEURONES 731 Alderlin, and to a lesser extent chlorpromazine, dibenzyline and phentolamine, often mimicked the excitant actions of the catecholamines. Alderlin (40 na) had a marked excitant action on the cell shown in Fig. 10 0, which was also excited by NA (60 na). Phentolamine (50 na) had a weak excitant action, manifested as a slight increase in the rate of firing and an augmentation of the L-glutamate response. Alderlin excitation was frequently followed by a period of depression of cell excitability. This effect is clearly illustrated in Fig. 10C. The response to L-glutamate was almost completely abolished after the alderlin-induced discharge had terminated and recovery took a further 2j min. Dual effects were also observed with phentolamine, although this compound rarely excited as powerfully as NA and alderlin. D-INPEA and MJ 1999 block peripheral,f-type catecholamine receptors and are reputed to have low potencies as local anaesthetics (Lish, Weikel & Duncan, 1965; Murmann, Saccani-Guelfi & Gamba, 1966). Their actions on thalamic neurones were of some interest since they were the only two antagonists tested in a survey of catecholamine receptors in the spinal cord which did not depress cell excitability (Biscoe, Curtis & Ryall, 1966). Both compounds, however, depressed catecholamine sensitive neurones in the thalamus in a similar manner to the other antagonists. D-INPEA (the laevo isomer) was considerably more active than L-INPEA (the dextro isomer). The lack of repeatability of NA excitation made it difficult to ascertain whether any of the antagonists was effectively opposing the excitant actions of this catecholamine. D-INPEA, however, appeared to reduce the magnitude of NA excitation on some neurones. Iproniazid, a monoamine oxidase inhibitor The monoamine oxidase inhibitor, iproniazid (l-isonicotinyl-2-isopropyl-hydrazine phosphate) was tested in order to ascertain whether it potentiated the action of monoamines. It was applied to ten cells, of which seven were depressed by NA. Iproniazid had a depressant action on these NA sensitive cells but did not depress the other three neurones. On one cell it appeared to potentiate the effects of NA. The depressant effects of iproniazid are unlikely to have been due to a potentiation of endogenously released NA or 5-HT as recovery occurred within a few minutes, whereas the inhibition of monoamine oxidase has a long duration. A direct action of iproniazid on receptors of thalamic neurones is more likely. 5-Hydroxytryptamine and related compounds 5-HT was tested on fifty-seven thalamic neurones. The results are summarized in Table 3. 5-HT was remarkable in that it had an effect on

18 732 J. W. PHILLIS AND A. K. TEBECIS most of the neurones on which it was tested. Its actions were comparable to those of the catecholamines in that it depressed the majority of the cells in the superficial group and excited a greater proportion of the deeper cells. Some of the actions of 5-HT are illustrated in Fig HT induced depression of L-glutamate firing is recorded in Fig. 11 A and the depression of a spontaneously firing unit in Fig. 11 B. Recovery was characteristically rapid. Other neurones were excited and there was a tendency for successive applications to have increasingly less effect. T.ABLE 3. Actions of 5-hydroxytryptamine on thalamic neurones Action Superficial Intermediate Deep No effect 3 (110) 1 (6%) 0 Depression 24 (86) 15 (83%) 8 (73%) Excitation 1 (3%) 2 (11%) 3 (27%) A 5-HT 20 5-HT 40 5-HT B ' ~~~~~~~ C ~ ~~ ooj ~~~~~~ 31X 5-HT 40 G 20 5-HT I A~~~~~~~~~~~~~~0 5.-HT 40 G 20 5-HT 40 5-HT 40 G 20, Fig. 11. Inhibitory and excitatory effects of 5-HT. A (5430 u): L-glutamate (40 na) firing was slightly depressed by 5-HT (20 na), markedly depressed by 5-1HT (40 na) and almost completely blocked by 5-HT (80 na). Recovery was typically rapid. B (4960,u): this unit was firing spontaneously. 5-HT (40 na) completely depressed this firing and recovery was rapid after termination of the drug. C (3970,u): L-glutamate (20 na) induced rapid firing. 5-HT (40 na), on two subsequent applications, induced marked firing with a longer latency of onset and offset. Both the depressant and excitant potencies of 5-HT in comparison with the catecholamines varied from neurone to neurone. Examples of 5-HT and catecholamine depressions are presented in Fig. 12. The neurone in Fig. 12A was discharging spontaneously and the passage of a positive current of 40 na through the recording barrel had little effect on this firing. Dopamine min E

19 MONOAMINES ON THALAMIC NEURONES 733 and NA (both 30 na) caused an appreciable reduction in the rate of firing, but their effects were not as marked as those of 5-HT (30 na) which abolished the spontaneous discharge. On another neurone, tested in the same animal with the same electrode as the last cell, 5-HT (20 na) was very much less effective than NA (20 na) and dopamine (20 na) in depressing L-glutamate firing. On other NA depressed cells, 5-HT had negligible actions and vice versa. It also failed to parallel the excitant actions of the catecholaimines; for example, 5-HT (50 na) depressed a thalamocortical neurone which was excited by NA (40 na) (Fig. 13). A Na+ 40 DA 30 5-HT 30 NA 30 B DA 20 5-HT 20 NA u In =1 _E _ u E 2 min Fig. 12. A comparison of depressant effects of DA, 5-HT and NA on two thalamic neurones. A (4090 gz): spontaneously firing unit which was virtually unaffected by Na+ (40 na) but markedly depressed by DA (30 na), 5-HT (30 na) and NA (30 na). 5-HT was more potent than DA or NA. B (740 /z): DA (20 na) 5-HT (20 na) and NA (20 na) all depressed L-glutamate (20 na) induced firing but on this unit DA and NA were more potent than 5-HT. The 5-HT antagonists, lysergic acid diethylamide (LSD) and methysergide, depressed most of the thalamic neurones upon which they were tested. These included neurones which were also depressed by 5-HT and some which were not affected by the latter in the amounts applied. An example of the latter type of unit is shown in Fig. 14A. 5-HT (60 na) 0

20 734 A J. W. PHILLIS AND A. K. TEBECIS B 5-HT 50 NA _~ ~~~100 u 03 I I I I 1 a min Fig. 13. An example of a NA-excited cell which was depressed by 5-HT, recorded at a depth of 6760 /t. A: antidromic spike evoked by stimulation of the ipsi-lateral cortex. B: 5-HT (50 na) depressed firing induced by L-glutamate (20 na) followed by a rapid recovery. NA (40 na) potentiated the response to L-glutamate, and increased rate of background firing. A 5-HT 60 LSD 60 I I I I I min I min _ 100 u E B Bufotenine 25 5-HT 25 5-HT 50 u 0) (A E min Fig. 14. A comparison ofdepressant effects of 5-HT, LSD and bufotenine. A (3860,u): 5-HT (60 na) produced no significant change in L-glutamate (60 na) induced firing but LSD (60 na) caused a depression which had a slow onset and a slow recovery. The latter response was typical of LSD. Chlorpromazine (40 na) and DA (40 na) had no effect on this unit. B (2440 #): bufotenine (25 na) caused a slow but pronounced depression of L-glutamate (40 na) firing but 5-HT (25 na) had no effect. 5-HT (50 na) caused some depression ofl-glutamate (40 na) firing but not as marked as bufotenine (25 na).

21 MONOAMINES ON THALAMIC NEURONES 735 failed to reduce the firing frequency evoked by L-glutamate, although LSD caused a marked reduction in the excitability of the neurone. Because of the magnitude and duration of the effects of LSD, it was difficult to ascertain whether it also antagonized the actions of 5-HT. Although 5-HT and LSD or methysergide were tested in conjunction on several cells, it was never possible to demonstrate any specific antagonism between them. Bufotenine is a more potent depressant of lateral geniculate nucleus neurones than 5-HT (Curtis & Davis, 1962) and it was found to be more effective in the thalamus as well. A sequence of responses to bufotenine and 5-HT is shown in Fig. 14B, Bufotenine (25 na) almost abolished the L-glutamate response, 5-HT (25 na) had no effect and 5-HT (50 na) caused some depression. Possible, monoamineryic pathway to the thalamus Fluorescent histochemical techniques have been used to map out a number of ascending monoamine neurone systems from the brain stem to the diencephalon (Anden et al. 1966). Noradrenergic and 5-hydroxytryptaminergic neurones, with their cell bodies situated in the pons and medulla oblongata project to the hypothalamus, thalamus and neocortex. Stimulation of the reticular formation was therefore attempted in several animals, whilst the excitability of thalamic neurones was monitored by the application of pulses of ACh or L-glutamate. In the absence of any effective specific antagonists for NA and 5-HT, it is not possible to draw any definite conclusions from these results concerning the involvement of these compounds as neurotransmitters in the thalamus. The findings strongly suggest that NA and 5-HT are involved in synaptic transmission in this area and confirmation of this hypothesis should be possible with the development of neural antagonists for these substances. Stimulation in the reticular formation inhibited some thalamic neurones and excited others. An example of a cell which was inhibited by both reticular formation stimulation and NA is shown in Fig. 15A. This cell was a member of the superficial group and like many other neurones in this region was strongly inhibited by reticular stimulation. Recovery after stimulation had ceased took approximately 2 min. Other cells frequently remained in a depressed state for several minutes. The neurone in Fig. 15 B was located in the ventrobasal complex and responded to reticular stimulation with a facilitated L-glutamate evoked discharge. During the application of L-glutamate the cell also responded with a long latency synaptic discharge during reticular stimulation, but the excitation was subthreshold in the non-glutamate-facilitated neurone. NA (40 na) also increased the excitability of the cell, causing a comparable increase in the glutamate response to reticular formation stimulation. 47 Physiol. I92

22 736 J. W. PHILLIS AND A. K. TEBECIS The effects of intravenously administered strychnine and picrotoxin were determined on monoamine and reticular formation induced depression of thalamic neurones. Strychnine (up to 1 mg/kg) had no effect on monoamine induced depressions of these cells. This finding is consistent with that A R.F. stimulation 1 0/sec NA u 0I 200 -( X. E 0 B R.F. stimulation 5/sec NA u VI 0. E 0 J 30 sec Fig. 15. Effects of reticular formation (R. F.) stimulation and NA. A (1270,u): R.F. stimulation (10/sec) depressed firing induced by L-glutamate (60 na). NA (20 na) blocked the response to L-glutamate. In another cell (B, 6890,t), R.F. stimulation (5/sec) potentiated firing to L-glutamate (50 na) and evoked a synaptic discharge. NA (40 na) did not fire this cell directly but markedly potentiated firing to L-glutamate (50 na). of Andersen, Eccles, Loyning & Voorhoeve (1963), who have shown that strychnine-resistant post-synaptic inhibitions are generated in thalamic neurones by stimulation of peripheral nerves. Picrotoxin has recently been shown to abolish strychnine-resistant post-synaptic inhibitions in the feline spinal cord (Kellerth & Szumski.

23 MONOAMINES ON THALAMIC NEURONES ). It also antagonized the depressant actions of noradrenaline and reticular formation stimulation on thalamic neurones. An example of this is shown in Fig. 16. L-glutamate excitation of this neurone was depressed by NA (40 na) and reticular stimulation (Fig. 16A, B). Picrotoxin (1 mg/kg) was administered intravenously and the responses in Fig. 16C recorded a few minutes subsequently. The effects of both NA and reticular stimulation were abolished. Picrotoxin also reduced or abolished the depressant effects of NA and reticular formation stimulation on the other thalamic neurones tested. A NA u a) R.F. stimulation NA 40 5/sec B R.F. C _ stimulation 5/sec sec Fig. 16. Inhibitory effects of intravenous picrotoxin on depression induced by NA and stimulation of reticular formation on a thalamic cell in the superficial layer. A: NA (40 na) caused an almost complete depression of firing induced by L-glutamate (40 na) and recovery took several mmn. B: R.F. stimulation (5/sec) also caused a complete depression of L-glutamate (40 na) firing. C: records of the same cell 2 min after an intravenous injection of picrotoxin (1 mg/kg). NA (40 na) and R.F. stimulation (5/sec) had no depressant action on L-glutamate (40 na) firing. The facilitatory effects of reticular formation stimulation on some cells are likely to be the result of ACh release as they can be blocked by ACh antagonists. Details of our studies on the cholinergic pathways to the thalamus will be described in a separate paper, but they appear to be similar to those which have been identified in the lateral geniculate nucleus (Phillis et at b). Facilitatory responses to reticular formation stimulation which were unaffected by ACh antagonists have also been observed. Different transmitters must also be involved in this pathway and the 47-2

24 738 J. W. PHILLIS AND A. K. TEBECIS results presented in this paper indicate that NA and 5-HT are candidates for this role. DISCUSSION The characteristics of monoamine receptors in the thalamus are clearly different from those of peripheral adrenergic receptors. The latter have been divided into two categories (cx- and f-receptors) on the basis of their responses to a variety of sympathomimetic amines (Ahlquist, 1948), and this has greatly simplified understanding of the actions of these compounds. Adrenaline is the most potent of the amines on a-receptors, with isoprenaline having least effect. Isoprenaline is the most potent on fl-receptors and noradrenaline the least effective. In general, effects on a-receptors are excitatory and those on fl-receptors inhibitory. The adrenergic blocking drugs are selective in their actions on peripheral receptors, some acting only on ac-receptors (e.g. dibenzyline and phentolamine) and others on,f-receptors (e.g. alderlin, D-INPEA and MJ 1999). So selective are these blocking agents that it has become customary to identify the type of receptor on the basis of the effect of blocking drugs on the responses to sympathomimetic amines (Innes & Nickerson, 1965; Nickerson, 1965). With the exception of dopamine, which depressed most of the cells tested, catecholamines depressed some cells, excited others and had no demonstrable effects on a third group. There did not appear to be any significant differences between the percentages of cells depressed by adrenaline, NA and isoprenaline. Although NA excited a greater proportion of neurones in the ventrobasal complex (the differences between NA and adrenaline are statistically significant) this result may have been influenced to an unpredictable extent by the marked inter-cat variation that occurred. These findings are difficult to reconcile with an Ahlquist-type classification, since, with the exception of dopamine, none of the sympathomimetic amines was significantly more potent as either a depressant or excitant than the others. The conclusion that thalamic receptors for sympathomimetic amines differ from peripheral receptors was supported by the failure of adrenergic antagonists to display any selectivity in their actions, as depression or excitation was observed when both ac- and fl-blockers were applied to thalamic neurones. The finding that NA had a dual action on some cells, initial applications causing excitation and later applications depression, can be explained by postulating that the excitatory receptors were desensitized by repeated drug applications revealing a depressant action. Implicit in this postulate is the assumption that some thalamic neurones possess at least two types of adrenergic receptor, and this could account for the diversity of effects of catecholamines on neurones of a uniform type (e.g. thalamocortical neurones) which were either depressed, excited or

25 MONOAMINES ON THALAMIC NEURONES 739 unaffected by these substances. A comparable experimental situation is found at the mammalian superior cervical ganglion, where it has been established, by the use of specific adrenergic antagonists, that there are a- and fl-receptors which mediate inhibition and excitation respectively (De Groat & Volle, 1966). Before the availability of these antagonists, however, numerous confficting reports appeared concerning the actions of catecholamines on sympathetic ganglia (Rothballer, 1959; Trendelenburg, 1961). The more marked excitant actions of NA and adrenaline on some thalamocortical neurones than others may have been the result of a greater proportion of available excitatory receptors on these particular cells. The presence of two types of catecholamine receptor on the same neurone would eliminate the necessity for postulating that excitation and depression are mediated through the same type of receptor. For instance, Oomura (1963) has suggested that the depolarizing action of ACh on some neurones and hyperpolarizing action on others in the mollusc, Onchidium, is due to an increase in Cl- ion permeability of the membrane of these cells. Depolarization or hyperpolarization would occur according as to whether the equilibrium potential for Cl- ion was below or above the membrane potential. The ACh receptor would be similar on both types of cell. However, an increased permeability to Cl- ion could not explain both effects of catecholamines on some thalamic neurones. The depressant effects of catecholamines on thalamic neurones are probably the result of increases in the membrane permeability to chloride and/or potassium ions. Synaptically evoked inhibitions in the thalamus are hyperpolarizing (Andersen, Eccles & Sears, 1964; Purpura, Frigyesi, McMurtry & Scarff, 1966), suggesting that potassium ions are involved in the synaptic processes. If the catecholamines are acting in a similar manner (which is likely, as they are also blocked by picrotoxin) it is reasonable to postulate that they also increase membrane permeability to potassium. An alternative explanation for the depressant actions of these compounds would be a presynaptic action, stimulating the release of transmitter from the terminals of inhibitory nerves. Whilst this possibility cannot be excluded, it appears to be less feasible than that of a postsynaptic locus of action. Catecholamine excitation can also be explained by assuming either preor post-synaptic sites of action and once again an action on the post-synaptic cell appears to be most likely. In the latter instance, depolarization could be due either to an increase in sodium permeability of the membrane (by itself or in conjunction with increases in permeability to smaller ions as well) or to a decrease in potassium permeability. Either process would result in a decreased membrane potential and consequent increase in cell excitability. A comparison of the results obtained with dopamine and those of the

26 740 J. W. PHILLIS AND A. K. TEBECIS other catecholamines reveals that DA depressed a significantly greater proportion of neurones on which it was tested and was devoid of excitant activity. The characteristically short duration of action of DA also distinguishes it from the other catecholamines, suggesting that it may be acting on a different type of receptor. The adrenergic antagonists had depressant actions on some thalamic neurones and many of them also mimicked the excitant actions of adrenaline and NA. The antagonists included some with actions at peripheral ac-receptor sites (dibenzyline phentolamine and chlorpromazine; Nickerson, 1965) and others with actions at fl-receptor sites (alderlin, D-INPEA and MJ 1999; Nickerson, 1965; Lish et al. 1965). A membrane stabilizing action of some of these compounds has previously been described and their depressant actions on interneurones in the spinal cord attributed to this action; Gill & Vaughan Williams, 1964; Biscoe et al. 1966). MJ 1999 and D-INPEA were of especial interest, as they have low potencies as local anaesthetics (Lish et al. 1965; Murmann et al. 1966) and did not depress the excitability of spinal cord interneurones (Biscoe et al. 1966). The depressant actions of these two substances on thalamic neurones suggests that they, and probably also the other antagonists, were themselves acting as sympathomimetic agents on the adrenergic receptors. This conclusion is supported by the finding that, in parallel with adrenaline and NA, the adrenergic antagonists did not depress all the cells upon which they were tested as would have been anticipated if they had been acting as membrane stabilizing agents. D-INPEA, which is considerably more active than L-INPEA as a fl-blocker at peripheral receptors (Almirante & Murmann, 1966), was the more potent depressant of thalamic neurones. The potent depressant actions of the adrenergic antagonists rendered them useless as blockers of NA depression and unsuitable for attempting to estimate whether neural inhibition was adrenergically mediated. With the possible exceptions of MJ 1999 and D-INPEA (which were not as extensively tested as some of the other substances) all the antagonists excited some thalamic neurones. Alderlin was the most potent excitant in this group, sometimes exceeding NA and adrenaline in potency. Alderlin excitation was frequently followed by a period during which the cell was depressed, testifying again to the presence of a dual receptor system on the membrane of some cells. D-INPEA was the only compound which appeared to reduce the magnitude of NA induced excitation. However, due to the rapidity with which NA excitation declined with repeated applications it was difficult to be certain of this finding. For the same reason, it was not possible to confirm a report that chlorpromazine selectively depresses NA induced excitation of neurones as has been recorded in the brain stem (Bradley, Wolstencroft, H6sli & Avanzino, 1966).

27 MONOAMINES ON THALAMIC NEURONES HT had both depressant and excitant actions on thalamic neurones; and although it frequently had the same action as NA or adrenaline on a given cell this was not invariably so. It is possible therefore that 5-HT acts on specific receptors which are not identical with those that react with the catecholamines. A weak depressant action of 5-HT on neurones in the ventro-basal thalamus has previously been described (Andersen & Curtis, 1964). It depressed amino acid and ACh induced firing of thalamic neurones in pentobarbitone sodium anaesthetized cats as well as spontaneous spindle activity but did not depress synaptic firing. The present series of experiments has confirmed its actions on amino acid, ACh and spontaneous firing and in addition it has been shown to depress the monosynaptic response evoked by stimulation of the brachium conjunctivum. LSD and methysergide depressed most of the neurones on which they were tested. A membrane stabilizing action of these compounds has been described (Di Carlo, 1961; Krnjevi6 & Phillis, 1963c) and this may account for their actions in the thalamus. Because of the duration of the depressant effects of these compounds it was difficult to ascertain whether they antagonized the actions of 5-HT. The depressant actions of the monoamines on thalamic neurones are comparable to those that were observed in the lateral geniculate nucleus (Phillis et al a) and the possibility that they act as neurotransmitters in both areas arises. The biochemical and histochemical evidence for a role of NA and 5-HT in synaptic transmission in the thalamus was described in the introduction to this paper. An ascending monoaminergic projection system from the brain stem to the diencephalon and telencephalon has recently been described (Anden et al. 1966) and this probably constitutes the major source of monoamine containing nerve fibres to the thalamus. The finding that picrotoxin reduced or abolished the depressant effects of NA and reticular formation stimulation is of considerable significance. The mechanisms ofaction ofpictotoxin are little understood. Umrath (1953) has suggested that it inhibits an enzyme which destroys an excitatory transmitter substance and in this manner produces an excess of excitation with ensuing convulsive activity. It is also believed that picrotoxin blocks presynaptic inhibition in the spinal cord (Eccles, Schmidt & Willis, 1963). It has been reported that picrotoxin does not influence the inhibition of Purkinje cells in the cerebellum (Crawford, Curtis, Voorhoeve & Wilson, 1963) or inhibitions in the spinal cord (Curtis, 1963). Kellerth & Szumski (1966), on the other hand, were able to show that picrotoxin abolished strychnine resistant inhibitions of spinal cord motoneurones and its ability to block several types of inhibitory junction in invertebrates is well documented (Robbins & Van der Kloot, 1958; Robbins 1959; Grundfest, Reuben & Rickles, 1959; Usherwood & Grundfest, 1964).

28 742 J. W. PHILLIS AND A. K. TEBECIS Picrotoxin also blocks the inhibitory actions of y-aminobutyric acid (GABA) at crustacean neuromuscular junctions. Considerable evidence exists to indicate that GABA is an inhibitory transmitter at such junctions. The reduction of inhibitory effects of reticular formation stimulation by picrotoxin could be the result either of an action on presynaptic terminals and resultant decrease in the release of inhibitory transmitter or of an action on the post-synaptic cell. As it also blocked NA depression, it appears that a major component of its action was on the post-synaptic cell. Picrotoxin block of post-synaptic inhibitions could result from at least two different actions. Attachment of the picrotoxin molecule to receptor sites for the inhibitory transmitter would prevent the latter from triggering the permeability changes involved in its action. Alternatively, picrotoxin could be acting on the mechanism which alters membrane permeability as a consequence of the attachment of inhibitory transmitter to its receptors. In either instance, the fact that NA depression is blocked by picrotoxin suggests a close similarity between the actions of the inhibitory transmitter and the catecholamines. Further evidence of the relationship between them may be forthcoming when effective specific antagonists for catecholamine depression are developed and their effects on neural inhibition ascertained. NA has depressant effects on spinal motoneurones and Renshaw cells (Engberg & Ryall, 1966; Biscoe & Curtis, 1966), its actions on the latter being insensitive to strychnine. The presence of NA and 5-HT containing nerve terminals in the ventral horn of the spinal cord which make intimate contact with motoneurones has been described. It has been suggested that these may be part of a monosynaptic monoaminergic pathway from the reticular formation and medulla oblongata to motoneurones (Dahlstrom & Fuxe, 1965). It is possible that the strychnine resistant, picrotoxin sensitive inhibitions described by Kellerth & Szumski (1966) were adrenergic in nature. NA and 5-HT excited many neurones in the ventrolateral complex of the thalamus and the possibility that they also act as excitatory transmitters must be considered. The facilitatory action of reticular formation stimulation on some thalamic neurones was reduced or abolished by ACh antagonists (Phillis & Tebecis, 1967), suggesting that a cholinergic projection extends from the brain stem to the thalamus. Such a link has been postulated by Shute & Lewis (1963) on the basis of extensive histochemical studies on the distribution of acetylcholinesterase. The facilitatory effects of reticular formation stimulation on other cells was not affected by ACh antagonists, however, and the possibility that either NA or 5-HT were involved remains to be investigated.

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